(133 days)
The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit is intended as a qualitative test for the detection of IgM antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing by one of the following methods:
· Standard two-tier test methodology (STTT) using an IgM blot test following current interpretation guidelines, OR
• Modified two-tier test methodology (MTTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/ IgM ELISA Test.
The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test as the first-tier screening test.
Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
The kit includes 12 x 8 well Antigen Coated strips. Conjugate. Substrate, Stop Solution, Wash Buffer, Diluent, Negative Control, Positive Control, and Cutoff Control. The controls are provided to determine if the assay is functioning properly and to determine the antibody level. The reagents are sufficient for 96 determinations.
During the test procedure, antibodies to B. burgdorferi (sensu stricto) if present in the human serum sample will bind to the antigens coated onto the wells forming antigen-antibody complexes. Excess antibodies are removed by washing. A conjugate of goat anti-human IgM antibodies conjugated with horseradish peroxidase is then added, which binds to the antigenantibody complexes. Excess conjugate is removed by washing. This is followed by the addition of a chromogenic substrate, tetramethylbenzidine (TMB). If specific antibodies to the antigen are present in the patients' serum, a blue color will develop. The enzymatic reaction is then stopped with a stopping solution causing the contents of the well to turn yellow. The wells are read photometrically with a microplate reader at 450nm.
The antigens used in the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test kit is a combination of B. burgdorferi sensu stricto strain B31 lysate, B. burgdorferi sensu stricto strain 2591 lysate, and a recombinant VlsE from B. burgdorferi sensu stricto strain B31. The lysates use spirochetes growing in BSK-H complete medium until mid-exponential phase. The recombinant VlsE protein is produced in E. coli SURE2 cells and purified by affinity chromatography. The purity of each antigen is assayed by SDS-PAGE followed by Coomassie staining and/or Western blotting.
Here's an analysis of the provided text to extract the acceptance criteria and study details for the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit.
It's important to note that this document describes an In Vitro Diagnostic (IVD) device, not an AI/ML-based medical device. Therefore, many of the typical acceptance criteria and study designs associated with AI/ML, such as MRMC studies, expert adjudication, separate training/test sets with established ground truth protocols, and specific ground truth types (pathology, outcomes data, expert consensus), are not applicable or described in the same manner.
Instead, the acceptance criteria for IVDs typically revolve around analytical performance (sensitivity, specificity, precision, reproducibility, cross-reactivity, interference) and clinical concordance with a predicate device or clinical diagnosis.
Acceptance Criteria and Device Performance (IVD Device)
1. Table of Acceptance Criteria and Reported Device Performance
Since this is an IVD device, not an AI/ML device, the acceptance criteria are generally based on meeting certain performance metrics (e.g., specificity, sensitivity, precision, agreement with a predicate device) that demonstrate its substantial equivalence to a legally marketed device. The document does not explicitly state numerical acceptance thresholds for each metric, but rather presents the performance observed and implies that these results were deemed acceptable for market clearance.
Here's a table summarizing the reported device performance, which implicitly represents the met acceptance criteria for this IVD:
| Metric (Implicit Acceptance Criteria) | Reported Device Performance (Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA) | Context/Notes |
|---|---|---|
| Primary Comparison (STTT) | Comparison with predicate device Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test Kit (K894293) for Standard Two-Tier Test (STTT) methodology. | |
| Positive Percent Agreement (PPA) | 90.3% (93/103) with 95% CI (82.9% - 95.5%) | Against the Predicate IgM ELISA from prospective samples. |
| Negative Percent Agreement (NPA) | 99.6% (460/462) with 95% CI (98.5% - 99.9%) | Against the Predicate IgM ELISA from prospective samples. |
| Second-Tier PPA (against IgM blot) | 98.3% (58/59) with 95% CI (90.9% - 99.9%) | When Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test positive/equivocal samples were re-tested with an FDA-cleared IgM blot assay (as the second-tier for STTT). |
| Clinical Sensitivity (STTT) | Early: 75.9% (44/58)Disseminated: 100.0% (17/17)Late: 89.7% (35/39) | Compared to predicate IgM ELISA in clinically characterized samples. |
| CDC Panel Agreement (STTT) | Healthy: 93.0%Early Lyme: 76.7%Cardiac Lyme: 66.7%Neurological Lyme: 100.0%Late: 85.0%Look-alike disease: 81.1% | Agreement with clinical diagnosis for CDC panel. |
| Analytical Specificity | Endemic Region: 96.1%Non-endemic Region: 90.5% | Determined by testing 208 asymptomatic individuals. |
| Precision (Within-lab) | CV ranges from 1.3% to 13.3% depending on sample and parameter (SD or CV) | For various control and sample types across 12 days, duplicates per day. Lowest CV (positive control, SD) 1.3%, highest CV (negative sample, SD) 13.3%. |
| Reproducibility (Multi-site) | CV ranges from 2.5% to 18.6% depending on sample and parameter (SD or CV) | For various control and sample types tested at 3 sites, triplicates, twice per day for 5 days. Lowest CV (moderate positive, between run) 2.5%, highest CV (negative sample, total) 18.6%. |
| Cross Reactivity | Varies by condition (e.g., Tick-borne Relapsing Fever: 0% positive, Ehrlichiosis: 60% positive) | Some cross-reactivity noted for certain conditions (Ehrlichiosis, Babesiosis, Leptospirosis, Varicella Zoster Virus). This is expected for serological assays and acknowledged by the need for second-tier testing. |
| Interfering Substances | None detected performance effect for tested substances (Albumin, Bilirubin, Cholesterol, Hemoglobin, Triglycerides) | Tested at recommended concentrations; no effect on performance. |
| Secondary Comparison (MTTT) | Comparison of Modified Two-Tier Test (MTTT) methodology (using this device + VlsE-OspC IgG/IgM ELISA) against Standard Two-Tier Test (STTT) using the predicate IgM blot. | |
| Positive Percent Agreement (PPA) | 100.0% with 95% CI (83.2% - 100.0%) | For overall MTTT vs. STTT results for all prospective samples. |
| Negative Percent Agreement (NPA) | 95.7% with 95% CI (93.4% - 97.3%) | For overall MTTT vs. STTT results for all prospective samples. |
| Clinical Sensitivity (MTTT) | Early: 62.9% (39/62)Disseminated: 100.0% (22/22)Late: 82.9% (34/41) | Compared to predicate STTT-IgM in clinically characterized samples. Notably higher for Late Lyme than predicate STTT-IgM (82.9% vs 22.0%). |
| CDC Panel Agreement (MTTT) | Healthy: 100.0%Early Lyme: 76.7%Cardiac Lyme: 66.7%Neurological Lyme: 100.0%Late: 80.0%Look-alike disease: 96.7% | Agreement with clinical diagnosis for CDC panel. Notably higher for Late Lyme than predicate STTT-IgM (80.0% vs 35.0%) and for Look-alike Disease (96.7% vs 97.8%). |
2. Sample Sampled Used for the Test Set and Data Provenance
- Determination of Assay Cutoff: 208 normal sera (103 from endemic region, 105 from non-endemic region). Provenance not explicitly stated but implies a mix of endemic/non-endemic populations, likely within the US.
- Precision (Test Set): A panel of 4 samples (negative, high negative, low positive, moderate positive) + kit controls. Tested in-house.
- Reproducibility (Test Set): A panel of 4 samples (negative, high negative, low positive, moderate positive) + kit controls. Tested at three different sites.
- Analytical Specificity (Test Set): 208 asymptomatic individuals' samples from endemic and non-endemic regions.
- Cross Reactivity (Test Set): 277 samples from serum vendors, confirmed positive for specific markers (e.g., Tick-borne Relapsing Fever IgM, Treponemal Infections, etc.).
- Interfering Substances (Test Set): 3 samples (high negative, equivocal, low positive) spiked with interferents.
- Clinical Studies (Comparison with Predicate Device - STTT):
- Prospective Samples: 531 serum samples.
- Provenance: Collected from three sites (one internal, two external reference laboratories) "using prospective samples submitted for Lyme serology testing". This implies real-world, prospectively collected, likely US-based clinical samples.
- Clinical Studies (Sensitivity Study - STTT): 114 clinically characterized samples (early, disseminated, late stages of Lyme disease). Provenance not explicitly stated.
- Clinical Studies (CDC Panel - STTT): 280 positive and negative specimens from the Center of Disease Control (CDC). These are a characterized reference panel. Provenance is CDC, likely multi-site or pooled.
- Clinical Studies (Method Comparison – MTTT IgM, Prospective Study):
- Prospective Samples: 481 serum samples for the initial screening. 68 positive and equivocal samples carried forward for second-tier testing.
- Provenance: Collected from three sites (one internal, two external reference laboratories) "using prospective samples submitted for Lyme serology testing". Implies real-world, prospectively collected, likely US-based clinical samples.
- Clinical Studies (Sensitivity Study - MTTT): 125 clinically characterized samples (early, disseminated, late stages of Lyme disease). Provenance not explicitly stated.
- Clinical Studies (CDC Reference Panel - MTTT): 280 positive and negative specimens from the Centers of Disease Control (CDC).
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
For IVD devices like this one, ground truth is typically established by:
- Consensus of clinical diagnosis for disease stages (clinically characterized samples).
- Confirmed status from reference laboratories or biological repositories (e.g., CDC panel, serum vendors for cross-reactivity).
- Comparison to a legally marketed predicate device (as done for the main comparative studies).
The document does not specify a number of experts or their qualifications for establishing ground truth for the test set. Ground truth is derived from the established clinical status of the samples (e.g., clinically characterized samples, CDC panel, confirmed positive from serum vendors). This is standard for IVD submissions, where the "ground truth" often relies on a combination of patient history, symptoms, other laboratory findings, and established reference panels or predicate device results, rather than a panel of independent human readers interpreting medical images.
4. Adjudication Method for the Test Set
Not applicable in the usual sense for an IVD kit. There's no human "reader" adjudication described. The "adjudication" is inherent in the established clinical status of the samples used as ground truth or the comparison to a predicate device. For instances where samples were "clinically characterized," this implies a clinical diagnosis that served as the reference standard, rather than an adjudication process of human interpretations of imaging/data.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance
Not Applicable. This is an IVD (laboratory diagnostic test kit), not an AI/ML-based medical imaging and interpretation device. MRMC studies are used for evaluating AI/ML systems that assist human readers in tasks like image interpretation.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done
Yes, in essence. The performance metrics (PPA, NPA, sensitivity, specificity, etc.) presented in the tables are the standalone performance of the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit itself, analogous to an "algorithm only" performance for an IVD. There is no "human-in-the-loop" component for the performance of this diagnostic test kit once it is run according to its instructions. The interpretation of the overall Lyme disease diagnosis, however, is intended to be made by a clinician based on multiple factors, as stated in the Indications for Use.
7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc)
The ground truth for this IVD was established using a combination of:
- Clinical Diagnosis / Clinically Characterized Samples: For the "Sensitivity Study" and "CDC Panel," samples were derived from patients with known clinical diagnoses of Lyme disease stages (early, disseminated, late) or from healthy individuals.
- Reference Panels: Specifically, the CDC Panel which consists of "positive and negative specimens... for Lyme disease detection" that are "masked characterized serum panel." This implies a very high confidence in the true status of these samples.
- Confirmed Status from Vendors: For the "Cross Reactivity" study, samples were "obtained from serum vendors who confirmed their positivity for each respective marker."
- Predicate Device/Established Methodology: For the "Comparison with Predicate Device" and "MTTT Comparison" studies, the performance was measured against results obtained from a legally marketed predicate device (Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test Kit) or an established "Standard two-tier test methodology (STTT)" using the predicate IgM blot test. While the predicate is not "ground truth" in the pathological sense, it serves as the reference standard for substantial equivalence studies for IVDs.
8. The Sample Size for the Training Set
Not applicable for this type of IVD device. This is a laboratory immunoassay kit, not an AI/ML algorithm that undergoes a distinct "training" phase with a large dataset. The "training" in the context of IVD development would be the R&D and optimization process to develop the assay components and parameters (e.g., antigen formulation, antibody concentrations, incubation times, cutoff determination), which are iterative and not typically reported as a "training set" size in an FDA submission. The determination of the assay cutoff involved 208 normal sera, which could be considered part of the assay's "calibration" or optimization, but not a "training set" in the sense of supervised machine learning.
9. How the Ground Truth for the Training Set was Established
Not applicable. As explained in point 8, there isn't a "training set" in the AI/ML context for this IVD device. The assay development and cutoff determination process is a different methodology. The "ground truth" for establishing the assay cutoff was based on 208 normal sera (from endemic and non-endemic regions) and a subsequent ROC analysis to optimize sensitivity and specificity.
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March 22, 2021
Gold Standard Diagnostics Jennifer Roth Vice President, Product Development 2851 Spafford St. Davis, California 95618
Re: K203295
Trade/Device Name: Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema Pallidum Treponemal Test Reagents Regulatory Class: Class II Product Code: LSR Dated: November 4, 2020 Received: November 9, 2020
Dear Jennifer Roth:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's
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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Maria Ines Garcia, Ph.D. Branch Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K203295
Device Name
Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit
Indications for Use (Describe)
The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit is intended as a qualitative test for the detection of IgM antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing by one of the following methods:
· Standard two-tier test methodology (STTT) using an IgM blot test following current interpretation guidelines, OR
• Modified two-tier test methodology (MTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/ IgM ELISA Test.
The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test as the first-tier screening test.
Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
| Type of Use (Select one or both, as applicable) | |
|---|---|
| ☑ Prescription Use (Part 21 CFR 801 Subpart D) | ☐ Over-The-Counter Use (21 CFR 801 Subpart C) |
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510(k) Summary
This 510(k) summary is being submitted in accordance with the requirement of SMDA 1990 and 21 CFR 807.92.
- Submitter's Name: Gold Standard Diagnostics Address: 620 Cantrill Drive Davis, CA. 95618 Phone Number: 530-759-8000 Contact Person: Jennifer Roth Date: November 3, 2020
-
- Product and Trade Name: Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit
Common Name: Lyme ELISA Test
- Product and Trade Name: Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit
Regulation Section:
(21 CFR 866.3830) Treponema pallidum treponemal test reagents.
Classification: Class II
Product Code: LSR; Reagent, Borrelia Serological Reagent
- Note: This clearance is for a modified use (Modified Two-tier Testing or MTTT use) for the previously cleared IVD test, the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit (K200023). The information and study data for the modified use is presented under the heading of "MTTT Comparison" below. With the exception of the intended use, all other information and data remain the same.
3) Legally Marketed Device to Which the Submitter Claims Equivalence:
- a. STTT Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test Kit (K894293).
- b. MTTT Gold Standard Diagnostics Borrelia burgdorferi IgM Blot Test Kit (K113846).
4) Description of the Device:
The kit includes 12 x 8 well Antigen Coated strips. Conjugate. Substrate, Stop Solution, Wash Buffer, Diluent, Negative Control, Positive Control, and Cutoff Control. The controls are provided to determine if the assay is functioning properly and to determine the antibody level. The reagents are sufficient for 96 determinations.
During the test procedure, antibodies to B. burgdorferi (sensu stricto) if present in the human serum sample will bind to the antigens coated onto the wells forming antigen-antibody complexes. Excess antibodies are removed by washing. A conjugate of goat anti-human IgM
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antibodies conjugated with horseradish peroxidase is then added, which binds to the antigenantibody complexes. Excess conjugate is removed by washing. This is followed by the addition of a chromogenic substrate, tetramethylbenzidine (TMB). If specific antibodies to the antigen are present in the patients' serum, a blue color will develop. The enzymatic reaction is then stopped with a stopping solution causing the contents of the well to turn yellow. The wells are read photometrically with a microplate reader at 450nm.
The antigens used in the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test kit is a combination of B. burgdorferi sensu stricto strain B31 lysate, B. burgdorferi sensu stricto strain 2591 lysate, and a recombinant VlsE from B. burgdorferi sensu stricto strain B31. The lysates use spirochetes growing in BSK-H complete medium until mid-exponential phase. The recombinant VlsE protein is produced in E. coli SURE2 cells and purified by affinity chromatography. The purity of each antigen is assayed by SDS-PAGE followed by Coomassie staining and/or Western blotting.
5) Intended Use of the Device:
The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit is intended as a qualitative test for the detection of IgM antibodies to B. burgdorferi sensu stricto in human serum from symptomatic patients or people suspected of infection. When used as the first-tier screening test, positive and equivocal results must be supplemented through additional testing bv one of the following methods:
- Standard two-tier test methodology (STTT) using an IgM blot test following current ● interpretation guidelines, OR
- . Modified two-tier test methodology (MTTT) using the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test.
The assay can also be used as a second-tier confirmation test using the MTTT methodology when used with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IzM ELISA Test as the first-tier screening test.
Positive test results by either the STTT or MTTT methodology are supportive evidence for the presence of antibodies and exposure to Borrelia burgdorferi, the cause of Lyme disease. A diagnosis of Lyme disease should be made based on the presence of Borrelia burgdorferi antibodies, history, symptoms, and other laboratory findings.
6) Comparison with the Predicate Device:
The tables below provide a comparison of the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test kit with the Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test kit (predicate device: K894293).
| Similarities | ||
|---|---|---|
| Item | Subject Device: Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit | Predicate Device: Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test Kit |
| Intended Use | The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA | Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test System is a |
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| Test kit is intended as a qualitativepresumptive (first step) test for thedetection of IgM antibodies to B.burgdorferi sensu stricto in humanserum from symptomatic patientsor people suspected of infection.Positive and equivocal resultsmust be supplemented by testingwith a second-step Western blotassay. | qualitative test intended for use in thepresumptive detection of human IgMantibodies to Borrelia burgdorferi inhuman serum. This EIA system shouldbe used to test serum from patients witha history and symptoms of infection withB. burdorferi. All positive and equivocalspecimens should be retested with ahighly specific, second-tier test such asWestern blot. Positive second-tierresults are supportive evidence ofinfection with B. burdorferi. Thediagnosis of Lyme disease should bemade based on history and symptoms(such as erythema migrans), and otherlaboratory data, in addition to thepresence of antibodies to B. burdorferi.Negative results (either first or second-tier) should not be used to exclude Lymedisease. | |
|---|---|---|
| Assay Format | Antigen coated microtiter plate -96 wells. | Same |
| Technology | ELISA | Same |
| Sample Matrix | Human serum | Same |
| Sample Processing | Dilute Samples 1:100 in Diluent | Same |
| Controls Provided | Positive, Cutoff, Negative | Same |
| Reagents Provided | Diluent, Wash, Conjugate,Substrate, Stop Solution,Absorption Solution | Same |
| Reported Results | Positive, Equivocal, Negative | Same |
| Interpretation | Optical density readings fromSpectrophotometer | Same |
| Differences | ||
|---|---|---|
| Item | Subject Device: Gold StandardDiagnostics Borrelia burgdorferiIgM ELISA Test Kit | Predicate Device: Trinity BiotechMarDx Borrelia burgdorferi EIAIgM Test Kit |
| Volumes | 100ul sample, 50ul substrate, 50ulstop solution | 100ul sample, 100ul substrate,100ul stop solution |
| Incubation | 15/15/15 minutes at roomtemperature | 30/30/10 minutes at roomtemperature |
| Antigens | B. burgdorferi B31 strain,B. burgdorferi 2591 strain,B. burgdorferi recombinant VlsE | B. burgdorferi B31 strain |
| Results Interpretation | Convert to units.Negative <9Equivocal 9.0-11.0 | Convert to units.Negative <0.80Equivocal 0.80-1.19 |
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| Positive >11.0 | Positive ≥1.2 | |
|---|---|---|
| -- | ---------------- | --------------- |
6(b1): Nonclinical Studies:
Determination of the Assav Cutoff
The cutoff was determined by testing a total of 208 normal sera which consisted of 103 sera from an endemic region of Lyme disease and 105 sera from a non-endemic region of Lyme disease. The mean plus two standard deviations was used to determine the assay cutoff. A known positive sample was then diluted to produce a ready to use cutoff control. An additional 194 samples consisting of 114 samples from different phases of Lyme disease, 8 negative healthy samples, 72 negative Lyme disease samples but do have other diseases that may cause serologic cross-reactivity, were tested. A receiver operating characteristics (ROC) analysis was performed to evaluate the performance of the assay and confirm that the chosen cutoff provided the best compromise between sensitivity and specificity.
Precision
To determine the precision of the Borrelia burgdorferi IgM ELISA Test, a within-lab precision study was conducted. A precision panel consisting of a negative sample, a high negative sample, a low positive sample, and a moderate positive sample, along with the kit controls, was tested in-house. Each of the panel members was tested in duplicate, twice per day, for 12 days. The sample panel was masked and randomized. The results are summarized in the following table:
| Sample | N | MeanUnits | Within-Run | Between-Run | Between-Day | Total | |
|---|---|---|---|---|---|---|---|
| ModeratePositive | 48 | 20.6 | SD | 1.234 | 0.476 | 0.423 | 1.222 |
| CV | 6.0% | 2.3% | 2.1% | 5.9% | |||
| LowPositive | 48 | 12.4 | SD | 0.849 | 0.728 | 0.405 | 0.834 |
| CV | 6.9% | 5.9% | 3.3% | 6.7% | |||
| HighNegative | 48 | 5.7 | SD | 0.740 | 0.495 | 0.349 | 0.727 |
| CV | 13.1% | 8.7% | 6.2% | 12.8% | |||
| Negative | 48 | 2.5 | SD | 0.328 | 0.103 | 0.061 | 0.324 |
| CV | 13.3% | 4.2% | 2.5% | 13.1% | |||
| PositiveControl | 48 | 25.2 | SD | 1.197 | 0.337 | 0.492 | 1.183 |
| CV | 1.3% | 1.3% | 2.0% | 4.7% | |||
| CutoffControl | 48 | 9.9 | SD | 0.317 | 0.238 | 0.305 | 0.287 |
| CV | 3.2% | 2.4% | 3.1% | 2.9% | |||
| NegativeControl | 48 | 0.7 | SD | 0.124 | 0.052 | 0.017 | 0.123 |
| CV | 18.1% | 736% | 2.5% | 17.9% |
Reproducibility
A reproducibility panel consisting of a negative sample, a high negative sample, a low positive sample, and a moderate positive sample, along with the kit controls, was tested at three
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different sites. The sample panel was masked and randomized. Each of the panel members was tested in triplicate, twice per day, for five days. The Within-Run, Between-Run, Between-Days, and Between-Sites Standard Deviation and Coefficients of Variation (CV) were calculated. The results are summarized in the following table:
| Sample | N | MeanUnits | Within-Run | Between-Run | Between-Day | Between-Site | Total | |
|---|---|---|---|---|---|---|---|---|
| ModeratePositive | 90 | 54.4 | SD | 3.26 | 1.37 | 2.49 | 1.38 | 3.73 |
| CV | 6.0% | 2.5% | 4.6% | 2.5% | 6.9% | |||
| LowPositive | 90 | 17.1 | SD | 1.13 | 0.46 | 0.98 | 0.78 | 1.29 |
| CV | 6.6% | 2.8% | 5.7% | 4.6% | 7.6% | |||
| HighNegative | 90 | 6.5 | SD | 0.60 | 0.34 | 0.48 | 0.56 | 0.75 |
| CV | 9.2% | 5.2% | 7.4% | 8.6% | 11.4% | |||
| Negative | 90 | 1.8 | SD | 0.31 | 0.25 | 0.29 | 0.32 | 0.34 |
| CV | 17.0% | 14.3% | 15.9% | 17.5% | 18.6% | |||
| PositiveControl | 30 | 24.2 | SD | 1.69 | 1.10 | 1.28 | 0.34 | 1.64 |
| CV | 7.0% | 7.0% | 5.3% | 3.5% | 6.8% | |||
| CutoffControl | 60 | 9.9 | SD | 0.36 | 0.17 | 0.22 | 1.33 | 0.34 |
| CV | 3.6% | 1.7% | 2.2% | 5.5% | 3.4% | |||
| NegativeControl | 30 | 0.7 | SD | 0.09 | 0.03 | 0.04 | 0.04 | 0.72 |
| CV | 13.3% | 5.1% | 5.1% | 5.0% | 13.1% |
Analytical Specificity
The analytical specificity was determined by testing 208 asymptomatic individuals' samples from endemic and non-endemic regions. The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test results are summarized in the following table:
| Number of Samples | Number Positive/Equivocal | Analytical Specificity | |
|---|---|---|---|
| Endemic Region | 103 | 4 | 96.1% |
| Non-endemic Region | 105 | 10 | 90.5% |
Cross Reactivity
A study using 277 samples was conducted to evaluate potential cross reactivity from different disease conditions. The samples were obtained from serum vendors who confirmed their positivity for each respective marker. The samples were tested on the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test. The results are summarized in the following table:
| Infection / Diagnosis | Number ofSera Tested | # Positive /(%) |
|---|---|---|
| Tick-borne Relapsing Fever IgM | 21 | 0 / (0%) |
| Treponemal Infections (TPPA) | 29 | 0 / (0%) |
| Rickettsia IgM | 23 | 0 / (0%) |
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| Ehrlichiosis IgM | 10 | 6 / (60%)* |
|---|---|---|
| Babesiosis IgM | 16 | 10 / (63%)* |
| Leptospirosis IgM | 10 | 8/ (80%)* |
| H. pylori IgM | 10 | 0 / (0%) |
| Epstein-Barr Virus IgM | 14 | 0 / (0%) |
| Varicella Zoster Virus | 16 | 6 / (38%) |
| Fibromyalgia | 25 | 0 / (0%) |
| Rheumatoid Arthritis | 12 | 0 / (0%) |
| Autoimmune Disease | 46 | 0 / (0%) |
| Multiple Sclerosis | 22 | 0 / (0%) |
| Severe Periodontitis | 23 | 0 / (0%) |
*Also positive on the predicate device
Interfering Substances
The effect of potential interfering substances on samples using the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test was evaluated. Three samples, a high negative, an equivocal and a low positive were spiked with high levels of interferants and were tested along with serum without spiked interferants. The recommended concentrations from the guideline "Interference Testing in Clinical Chemistry" EP07-A3 from the Clinical and Laboratory Standards Institute were used (see table below). The tested substances did not affect the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test.
| Substance | Concentration | Interference |
|---|---|---|
| Albumin | 60 mg/ml | None detected |
| Bilirubin | 0.4 mg/ml | None detected |
| Cholesterol | 4.0 mg/ml | None detected |
| Hemoglobin | 10 mg/ml | None detected |
| Triglycerides | 15 mg/ml | None detected |
6(b2): Clinical Studies:
Comparison with Predicate Device
Comparison studies were conducted at three sites (one internal and two external reference laboratories) using prospective samples submitted for Lyme serology testing. Five hundred thirty one (531) serum samples were tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test and on the predicate B. burgdorferi IgM ELISA Test. The results are summarized in the following table:
| Predicate IgM ELISA | |||||
|---|---|---|---|---|---|
| Positive | Equivocal* | Negative | Total | ||
| Gold Standard DiagnosticsBorrelia burgdorferi IgMELISA Test Kit | Positive | 72 | 9 | 5 | 86 |
| Equivocal* | 4 | 8 | 5 | 17 | |
| Negative | 1 | 9 | 418 | 428 | |
| Total | 77 | 26 | 428 | 531 |
*Equivocal samples counted as positive
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| Positive percent agreement = 90.3% (93/103) | 95% CI (82.9% - 95.5%) |
|---|---|
| Negative percent agreement = 99.6% (460/462) | 95% CI (98.5% - 99.9%) |
Second-Tier Testing
All positive and equivocal samples by the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test and by the Predicate IgM ELISA were tested by a FDA cleared IgM blot assay. The results are summarized in the following table:
| Tier 1 Positiveor Equivocal | IgM BlotPositive | IgM BlotNegative | |
|---|---|---|---|
| Predicate IgM ELISA | 103 | 59 | 44 |
| Gold StandardDiagnostics Borreliaburgdorferi IgMELISA Test Kit | 103 | 61 | 42 |
| Predicate IgM ELISA+Gold StandardDiagnostics Borreliaburgdorferi IgMELISA Test Kit | 93 | 58 | 35 |
| Percent Agreement with Predicate Device | ||
|---|---|---|
| 2nd Tier PPA(95% CI) | 98.3%(90.9% - 99.9%) | 58/59 |
Clinical Sensitivity
Sensitivity Study
A sensitivity study was performed on 114 clinically characterized samples. The samples encompass early, disseminated, and late stages of Lyme disease. The samples were tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test and on the predicate B. burgdorferi IgM ELISA Test. The results are summarized in the following table:
| DiseaseStage | n | Gold StandardDiagnostics Borreliaburgdorferi IgMELISA Test Kit | PredicateIgM ELISA |
|---|---|---|---|
| Early | 58 | 75.9% (44/58) | 77.6% (45/58) |
| Disseminated | 17 | 100.0% (17/17) | 100.0% (17/17) |
| Late | 39 | 89.7% (35/39) | 84.6% (33/39) |
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CDC Panel
A panel of 280 positive and negative specimens from the Center of Disease Control (CDC) for Lyme disease detection was tested on both the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test and on the predicate device. The results are presented as a means to convey further information on the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test with a masked characterized serum panel. The results are summarized in the following table:
| Gold Standard DiagnosticsBorrelia burgdorferi IgMELISA Test Kit | PredicateIgM ELISA | ||||
|---|---|---|---|---|---|
| Disease Stage | n | PositiveorEquivocal | % Agreementwith ClinicalDiagnosis | PositiveorEquivocal | % Agreementwith ClinicalDiagnosis |
| Healthy | 100 | 7 | 93.0% | 14 | 86.0% |
| Early Lyme | 60 | 46 | 76.7% | 48 | 80.0% |
| Cardiac Lyme | 3 | 2 | 66.7% | 2 | 66.7% |
| Neurological Lyme | 7 | 7 | 100.0% | 7 | 100.0% |
| Late | 20 | 17 | 85.0% | 17 | 85.0% |
| Look-alikeDisease | 90 | 17 | 81.1% | 25 | 72.2% |
Expected Values
The range of values and positivity rate among different studies and population for the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test are as follows:
| Unit Results | Qualitative Results | |||||
|---|---|---|---|---|---|---|
| Population | # Samples | Mean | Range | Std. Dev. | # Positive/ Equivocal | % Positive/ Equivocal |
| Normal Endemic | 103 | 3.5 | 0.4 - 10.7 | 2.341 | 4 | 3.9% |
| Normal Non-Endemic | 105 | 4.2 | 0.0 - 15.5 | 3.136 | 10 | 9.5% |
| Prospective Study | 531 | 7.7 | 0.0 - 76.6 | 11.742 | 103 | 19.4% |
| Sensitivity Study | 114 | 32.6 | 0.0 - 81.0 | 21.138 | 96 | 84.2% |
Note: It is recommended that each laboratory determine its own normal range based on the population.
7) Conclusion:
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From the comparison data, we conclude that the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test is substantially equivalent to the Trinity Biotech MarDx Borrelia burgdorferi EIA IgM Test kit (predicate device: K894293).
MTTT Comparison:
Comparison with the Predicate Device - MTTT:
The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test Kit, when used as the firststep or second-step test in combination with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test Kit in the Modified Two-tier Testing (MTTT) method, was compared to the Standard Two-tier testing (STTT) method using the predicates Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA (K200023) Test Kit as the first-step test followed by testing all the positive and equivocal results on the Gold Standard Diagnostics Borrelia burgdorferi IgM Blot Test (K113846). Below are tables comparing the two devices.
| Similarities | ||
|---|---|---|
| Item | Subject Device: Gold StandardDiagnostics Borrelia burgdorferi IgMELISA Test Kit | Predicate Device: Gold StandardDiagnostics Borrelia burgdorferiIgM Blot (K113846) |
| Intended Use | The Gold Standard Diagnostics Borreliaburgdorferi IgM ELISA Test Kit isintended as a qualitative test for thedetection of IgM antibodies to B.burgdorferi sensu stricto in humanserum from symptomatic patients orpeople suspected of infection. Whenused as the first-tier screening test,positive and equivocal results must besupplemented through additional testingby one of the following methods:•Standard two-tier test methodology(STTT) using an IgM blot test followingcurrent interpretation guidelines, OR•Modified two-tier test methodology(MTTT) using the Gold StandardDiagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test.The assay can also be used as a second-tier confirmation test using the MTTTmethodology when used with the GoldStandard Diagnostics Borrelia | The Gold Standard DiagnosticsBorrelia burgdorferi B31 IgG LineBlot Test Kit is intended for thequalitative detection of IgG antibodiesto B. burgdorferi sensu stricto (B31)in human serum. This test is intendedfor use in testing human serumsamples which have been foundpositive or equivocal using an ELISAor IFA test procedure to providesupportive evidence of infection withB. burgdorferi. |
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| burgdorferi VlsE-OspC IgG/IgMELISA Test as the first-tier screeningtest. | ||
|---|---|---|
| Positive test results by either the STTTor MTTT methodology are supportiveevidence for the presence of antibodiesand exposure to Borrelia burgdorferi ,the cause of Lyme disease. A diagnosisof Lyme disease should be made basedon the presence of Borrelia burgdorferiantibodies, history, symptoms, and otherlaboratory findings. | ||
| Antigens | B. burgdorferi B31 strain, | Same |
| Sample Matrix | Human serum | Same |
| Controls Provided | Positive, Cutoff, Negative | Same |
| Sample Processing | Dilute Samples 1:100 | Same |
| Assay Type | Qualitative | Same |
| Differences | ||
|---|---|---|
| Item | Subject Device: Gold StandardDiagnostics Borrelia burgdorferi IgMELISA Test Kit | Predicate Device: Gold StandardDiagnostics Borrelia burgdorferiIgM Blot (K113846) |
| Assay Format | Antigen coated microtiter plate – 96wells. | Nitrocellulose Strips |
| Reagents Provided | Diluent, Wash, Conjugate, Substrate,Stop Solution | Diluent/Wash, Conjugate, Substrate |
| Volumes | 100ul sample, 50ul substrate, 50ul stopsolution | 1500ul sample, 1500ul substrate, |
| Incubation | 15/15/15 minutes at room temperature | 30/30/10-13 minutes at roomtemperature |
| Interpretation | Optical density readings fromSpectrophotometer | Visual |
| ResultsInterpretation | Convert to units.Negative <9Equivocal 9.0-11.0Positive >11.0 | Compare to cutoff band |
| Reported Results | Positive, Equivocal, Negative | Positive, Negative |
Method Comparison Study – MTTT IgM
The following studies were conducted to determine the performance of the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test as a first-tier or second-tier assay in the modified two-tier testing (MTTT) methodology.
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Gold Standard Diagnostics MTTT-IgM ELISA Method Comparison: The Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test was utilized in a MTTT (2-ELISA) protocol with the Gold Standard Diagnostics Borrelia burgdorferi VlsE-OspC IgG/IgM ELISA Test. The MTTT (2-ELISA) results were compared to the standard two-tier testing (STTT) using the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA followed by testing all positive and equivocal results on the predicate IgM blot test.
Prospective Study
Comparison studies were conducted at three sites (one internal and two external reference laboratories) using prospective samples submitted for Lyme serology testing. Four hundred eighty-one (481) serum samples were tested on the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test. A total of 68 positive and equivocal samples were obtained.
In the STTT protocol the samples that were positive or equivocal (n=68) were tested with the predicate B. burgdorferi IgM blot test. In the MTTT protocol the samples (n=68) were tested on a second ELISA, the Gold Standard Diagnostics Borrelia burgdorferi VIsE-OspC IgG/IgM ELISA Test. In the second-tier ELISA test, positive and equivocal results were considered positive.
The results of the second-tier test of the STTT when compared to the second-tier test of the MTTT, including only the samples that were positive in the first tier, are summarized in the following table:
| Predicate IgM Immunoblot | ||||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| Gold Standard DiagnosticsBorrelia burgdorferi VlsE-OspC IgG/IgM ELISA | Positive | 20 | 20 | 40 |
| Negative | 0 | 28 | 28 | |
| Total | 20 | 48 | 68 |
Negative percent agreement = 58.3%
o C1 (83.2% - 100.0%) 95% CI (43.2% - 72.4%)
The results of the MTTT when compared to the STTT, including all samples that were part of the prospective study (n=481), are summarized in the following table:
| Predicate STTT- IgM | ||||
|---|---|---|---|---|
| Positive | Negative | Total | ||
| Gold Standard DiagnosticsMTTT- IgM | Positive | 20 | 20 | 40 |
| Negative | 0 | 441 | 441 | |
| Total | 20 | 461 | 481 |
Positive percent agreement = 100.0% Negative percent agreement = 95.7%
95% CI (83.2% - 100.0%) 95% CI (93.4% - 97.3%)
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Sensitivity Study
A sensitivity study was performed on 125 clinically characterized samples. The samples encompass early, disseminated, and late stages of Lyme disease. The samples were tested on both the Gold Standard Diagnostics MTTT-IgM and on the predicate STTT-IgM. The results are summarized in the following table:
| Gold Standard DiagnosticsMTTT – IgM | PredicateSTTT - IgM | ||||
|---|---|---|---|---|---|
| DiseaseStage | n | PositiveorEquivocal | % Agreementwith ClinicalDiagnosis | PositiveorEquivocal | % Agreementwith ClinicalDiagnosis |
| Early | 62 | 39 | 62.9% | 40 | 64.5% |
| Disseminated | 22 | 22 | 100.0% | 22 | 100.0% |
| Late | 41 | 34 | 82.9% | 9 | 22.0% |
CDC Reference Panel
A panel of 280 positive and negative specimens from the Centers of Disease Control (CDC) for Lyme disease detection was tested on both the Gold Standard Diagnostics MTTT-IgM and on the predicate STTT-IgM. The results are summarized in the following table:
| Disease Stage | n | Gold Standard DiagnosticsMTTT-IgM | Predicate STTT- IgM | ||
|---|---|---|---|---|---|
| PositiveorEquivocal | % Agreementwith ClinicalDiagnosis | PositiveorEquivocal | % Agreementwith ClinicalDiagnosis | ||
| Healthy | 100 | 0 | 100.0% | 1 | 99.0% |
| Early Lyme | 60 | 46 | 76.7% | 30 | 50.0% |
| Cardiac Lyme | 3 | 2 | 66.7% | 2 | 66.7% |
| Neurological Lyme | 7 | 7 | 100.0% | 7 | 100.0% |
| Late | 20 | 16 | 80.0% | 7 | 35.0% |
| Look-alike Disease* | 90 | 3 | 96.7% | 2 | 97.8% |
*infectious mononucleosis, fibromyalgia, multiple sclerosis, rheumatoid arthritis, syphilis and severe periodontitis
8) Conclusion:
From the comparison data, we conclude that the Gold Standard Diagnostics Borrelia burgdorferi IgM ELISA Test is substantially equivalent to the Gold Standard Diagnostics Borrelia burgdorferi IgM Blot Test Kit (K113846) when used for the Modified Two-tier Testing (MTTT) Lyme testing.
§ 866.3830
Treponema pallidum treponemal test reagents.(a)
Identification. Treponema pallidum treponemal test reagents are devices that consist of the antigens, antisera and all control reagents (standardized reagents with which test results are compared) which are derived from treponemal sources and that are used in the fluorescent treponemal antibody absorption test (FTA-ABS), theTreponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies toTreponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).